CN1380415A

CN1380415A - Synthesis of related gene for producing carotenoid in transgenic plant

Info

Publication number: CN1380415A
Application number: CN 01105878
Authority: CN
Inventors: 姚泉洪; 彭日荷; 熊爱生
Original assignee: BIOTECHNOLOGY RES CT SHANGHAI; Shanghai Yongye Agricultural Science Bioengineering Co ltd
Current assignee: BIOTECHNOLOGY RES CT SHANGHAI; Shanghai Yongye Agricultural Science Bioengineering Co ltd
Priority date: 2001-04-06
Filing date: 2001-04-06
Publication date: 2002-11-20

Abstract

The present invention relates to a related gene for proudcing carotenoid in transgenic plant. Said invention utilizes overlapping extension PCR amplification process and uses six genes of crtE, crtB, crtI, crtW, crtY and crtZ as template to synthesize the related gene for synthesizing astaxanthin with plant preference code and can make synthetic gene express in plant to produce the carotene substances of astaxanthin etc. so as to improve quality of grain crops and vegetable.

Description

Be used for genes involved synthetic that transgenic plant produce carotenoid

The present invention relates to the plant bioreactor in the plant genetic engineering field, more specifically say so by synthetic 6 genes involveds can in plant materials, producing carotenoid of the method for chemosynthesis.

Carotenoid is important natural pigment, extensively exists in the animal and plant body, and from having more than 600 kind of carotenoid to be found so far, its color is extremely orange red from yellow, the color that is used for modifying food for a long time.Along with the understanding to the carotenoid chemical structure, the function of carotenoid is excavated gradually.

Carotenoid has anti-oxidant function.9-11 conjugated double bond arranged in the carotenoid chemical structure, can with combined with radical.Cause cell senescence even death after avoiding the oxidative damage of cell to be accumulated to a certain degree.Carotenoid has anticancer change effect.In the influences of carotenoid to aflatoxin initiation liver cancer such as research astaxanthin, find that carotenoid can reduce the number and the size of liver lesion.Carotenoid has the enhancing body immunization.Carotenoid such as astaxanthin can stimulate production of antibodies (Liu Ziyi, 1997, Chinese marine drug).

The discovery of the numerous functions of carotenoid has increased on the market demand to this compounds greatly.Only astaxanthin is a kind of, and annual value of demand promptly surpasses 100,000,000 dollars (Liang Shizhong, 2000, food and fermentation industries).Present most of carotinoid compounds is to obtain by chemosynthesis, needs very high cost and resource (Johnson EA, 1995, Adv Biochem Eng Biotechnol).Cultivate Du Shi salt tolerant algae biology by big area and also can obtain a large amount of carotenoid, but this algae can only be in the breed of the area of arid, to the requirement of water quality, environment and light very high (Zheng Xiaodong, 2000, journal of Zhejiang university).Carotinoid compounds such as astaxanthin can also directly extract from first section class animal, but content is very low, are not suitable for mass production (Chinese feedstuff industry Information Network).Utilize carotinoid compounds such as microorganisms producing astaxanthin to become the emphasis of recent researches, wherein at most with red Fife's yeast research, this bacterium has some essential feature as the pigment biogenetic derivation: quick different oxygen metabolism, incubation time is short, can realize high density fermentation in fermentor tank.Though this quasi-microorganism has very big potentiality to be exploited, the defective that present ubiquity yields poorly also fails to commercially produce on a large scale (Wang Jufang, 2000, biotechnology progress).

Utilize genetic engineering technique, key gene clone and transformation with carotenoid such as synthesizing astaxanthins, utilize suitable promotor to regulate and control, these genes are transformed in bacterium, yeast and the plant simultaneously, realize efficiently expressing of carotinoid compounds such as astaxanthin, will satisfy the market that develops rapidly great demand these products.Wherein have more social value and marketable value with the plant of cultivating energy mass production β-Hu Luobusu and astaxanthin.As the golden paddy rice of material produce, golden wild cabbage and golden rape, will become the scenery of human health with crops such as paddy rice, wild cabbage and rapes.

Begin synthetic β-Hu Luobusu from glycolysis-product acetyl-CoA and need a series of enzyme catalysiss.Most of biologies will be through acetyl-CoA to farnesyl pyrophosphate (FPP) stage, and FPP synthesizes ergosterol or β-Hu Luobusu under different enzyme effects.From FPP to the β-Hu Luobusu, need four kinds of enzymes to participate in.Farnesyl pyrophosphate (FPP) is synthetic ridge ox base ridge ox base tetra-sodium (GGPP) under the catalysis of ridge ox base ridge ox base pyrophosphate synthetase (CrtE), GGPP produces phytoene under phytoene synthetase (CrtB) effect, under phytoene desaturase (CrtI) effect, form Lyeopene gradually afterwards, under lycopene beta cyclase (CrtY) catalysis, Lyeopene synthesizes β-Hu Luobusu at last.Astaxanthin is a β-Hu Luobusu synthetic terminating point, changes astaxanthin into by β-Hu Luobusu and need add two ketone groups and hydroxyl, external synthetic relatively difficulty.Can be under the catalysis of two enzymes but only need in vivo by the β-Hu Luobusu synthesizing astaxanthin.β-Hu Luobusu hydroxylase (CrtZ) and β-Hu Luobusu ketolase (CrtW) can add hydroxyl and ketone group on the C3 of β-Hu Luobusu C3 ' and C4 C4 ' position, form astaxanthin (Claudia SD, 2000, EnviromentalBiotechnology).

To so far, from bacterium, plant, algae and fungi, cloned nearly 150 respectively and synthesized relevant gene (Hirschberg J, 1997, Purelity Applied Chemical) with carotenoid.The gene that these functions are close is widely different on nucleotide level.On the one hand, these genes can be transformed in the different microorganism or plant according to function combinations together, produce various carotenoid products (Misawa N, 1998, Applied and Environmental Microbiology).On the other hand, the difference of gene provides assurance for plant production carotenoid product, the conversion of foreign gene can not change the function of congenerous gene in the plant, cause the gene silencing phenomenon, can strengthen the function of similar gene on the contrary, improve the output (Hirschberg J, 2000, Nature Biotechnology) of product.

The present invention is template (ncbi.nlm.nih.gov:80) with the amino acid of secondary coccus (Pracoccus marcusii) 6 kinds of relevant enzymes in astaxanthin is synthetic, according to the plant-preference password, and these genes of chemosynthesis.These 6 genes are respectively ridge ox base ridge ox base pyrophosphate synthetase gene (crtE); Phytoene synthase gene (crtB); Phytoene desaturase gene (crtI); Lycopene beta cyclase gene (crtY); β-Hu Luobusu '-hydroxylase gene (crtZ) and β-Hu Luobusu ketolase gene (crtW).

Most of carotenoid synthase genes (crt) clone and expression in intestinal bacteria once, but carotenoid output is extremely low in the intestinal bacteria (every gram dry cell weight only has the 10-500 microgram); With carotenoid synthase gene transformation yeast, can obtain higher output (every gram dry cell weight contains 0.4 milligram), be equivalent to the content astaxanthin (Johnson EA, 1995, Adv BiochemEng Biotechnol) that produces in red Fife's yeast.Yet most of yeast are easy to carry out an other pathways metabolism in the carotenoid building-up process, produce ergosterol, thereby the output of carotenoid is lowered (BalleyJE, 1991, Science).Utilize carotenoid materials such as plant production astaxanthin to have lot of advantages: (1) cost is low; (2) safety; (3) stable, can be hereditary, express the offspring.

Just because of have extremely tempting development prospect with plant production carotenoid, a lot of in the world laboratories are in the research work of carrying out this respect, and in tobacco, tomato, paddy rice, successfully synthesized the β-Hu Luobusu material, make the output of β-Hu Luobusu increase substantially raising (NormileD, 2000, Science).These research work have convincingly demonstrated plant has huge development potentiality in the exploitation carotinoid compounds.In order to improve the output of this compounds, we according to the plant-preference encryption design 6 genes of synthesizing astaxanthin.

The domestic report that does not also utilize gene engineering method to produce astaxanthin so far, once in food-yeast (Candida utilis), utilized above-mentioned 6 genes produce astaxanthins achieving success (MisawaN abroad, 1998, Applied and Environmental Microbiology), synthesizing astaxanthin also has successfully report in plant, but the intermediate product in the building-up process is the β-Hu Luobusu that plant self produces, because the content of β-Hu Luobusu in plant is very low, the also very low (BramleyPM of the content of astaxanthin, 2000, Nature Biotechnology).

The objective of the invention is to utilize overlapping extension PCR amplification technique is template with crtE, crtB, crtI, crtY, six genes of crtZ, crtW, synthetic astaxanthin synthesis related gene with plant-preference password.

Another object of the present invention is that the synthetic gene is expressed in plant, produces carotenoid materials such as astaxanthin.

A further object of the present invention is to utilize the synthetic gene to improve the quality of food crop and vegetables, helps the healthy protective foods of people with preparation.

The present invention adopts following method to implement the present invention:

Redesign the nucleotide sequence of beta carotene ketolase gene (CrtW) and lycopene beta cyclase gene (crtY) in the secondary coccus according to the plant-preference password, and form associating gene C rtWcrtY.

The overlapping extension PCR method of synthetic employing of CrtWcrtY gene carries out pcr amplification in conjunction with the high temperature resistant polysaccharase of high-fidelity.Nucleotide sequence is cloned and measured to gene in intestinal bacteria, be 1997bp Nucleotide from codon ATG to termination codon TAA length.Insert the preceding paragraph in 726～786 positions of gene and cut sequence automatically:

CAACTCCTCAACTTCGACCTCCTCAAACTCGCA

GGCGACGTCGAATCCAACCCAGGCCCA。

The Nucleotide and the aminoacid sequence of complete synthesis crtWcrtY gene are as follows:

ATGTCCGCACACGCACTCCCAAAAGCAGACCTCACCGCAACCTCCCTCATCGTCTCCGGTGGTATCATCGCA--?72

M S A H A L P K A D L T A T S L I V S G G I I A

GCATGGCTCGCACTCCACGTCCACGCACTCTGGTTCCTCGACGCAGCAGCACACCCAATCCTCGCAGTCGCA--144

A W L A L H V H A L W F L D A A A H P I L A V A

AACTTCCTCGGTCTCACCTGGCTCTCCGTCGGTCTCTTCATCATCGCACACGACGCAATGCACGGTTCCGTC--216

N F L G L T W L S V G L F I I A H D A M H G S V

GTCCCAGGTAGACCAAGAGCAAACGCAGCAATGGGTCAACTCGTCCTCTGGCTCTACGCAGGTTTCTCCTGG--288

V P G R P R A N A A M G Q L V L W L Y A G F S W

AGAAAGATGATCGTCAAGCACATGGCACACCACAGACACGCAGGTACTGACGACGACCCAGACTTCGACCAC--360

R K M I V K H M A H H R H A G T D D D P D F D H

GGTGGTCCAGTCAGATGGTACGCAAGATTCATCGGTACTTACTTCGGTTGGAGAGAAGGTCTCCTCCTCCCA--432

G G P V R W Y A R F I G T Y F G W R E G L L L P

GTCATCGTCACCGTCTACGCACTCATCCTCGGTGACAGATGGATGTACGTCGTCTTCTGGCCACTCCCATCC--504

V I V T V Y A L I L G D R W M Y V V F W P L P S

ATCCTCGCATCCATCCAACTCTTCGTCTTCGGCACCTGGCTCCCACACAGACCAGGTCACGACGCATTCCCA--576

I L A S I Q L F V F G T W L P H R P G H D A F P

GACAGACACAACGCAAGATCCTCCAGAATCTCCGACCCAGTCTCCCTCCTCACCTGCTTCCACTTCGGTGGC--648

D R H N A R S S R I S D P V S L L T C F H F G G

TACCACCACGAACACCACCTCCACCCAACCGTCCCTTGGTGGAGACTCCCATCCACCAGAACCAAAGGCGAC--720

Y H H E H H L H P T V P W W R L P S T R T K G D

ACCGCACAACTCCTCAACTTCGACCTCCTCAAACTCGCAGGCGACGTCGAATCCAACCCAGGCCCAATGACC--792

T A Q L L N F D L L K L A G D V E S N P G P M T

CACGACGTCCTCCTCGCAGGTGCAGGTCTCGCAAACGGTCTCATCGCACTCGCACTCAGAGCAGCAAGACCA--864

H D V L L A G A G L A N G L I A L A L R A A R P

GACCTCAGAGTCCTCCTCCTCGACCACGCAGCAGGTCCATCCGAAGGTCACACCTGGTCCTGCCACGACCCA--936

D L R V L L L D H A A G P S E G H T W S C H D P

GACCTCTCCCCACACTGGCTCGCAAGACTCAAGCCACTCAGAAGAGCAAACTGGCCAGACCAAGAAGTCAGA-1008

D L S P H W L A R L K P L R R A N W P D Q E V R

TTCCCAAGACACGCAAGAAGACTCGCAACCGGCTACGGCTCCCTCGACGGTGCAGCACTCGCAGACGCAGTC-1080

F P R H A R R L A T G Y G S L D G A A L A D A V

GTCAGATCCGGTGCAGAAATCAGATGGGACTCCGACATCGCACTCCTCGACGCACAAGGTGCAACCCTCTCC-1152

V R S G A E I R W D S D I A L L D A Q G A T L S

TGCGGTACTAGAATCGAAGCAGGCTCCGTCCTCGACGGCAGAGGCCCACACCCATCCAGACACCTCACCCTC-1224

C G T R I E A G S V L D G R G P H P S R H L T L

GGCTTCCACAAGTTCCTCGGTGTCGAAATCGAAACCGACAGACCACACGGTGTCCCAAGACCAGTCATCATG-1296

G F H K F L G V E I E T D R P H G V P R P V I M

GACGGCACCGTCACCCAAAGAGACGGCTACGGCTTCATCTACCTCCTCCCATTCTCCAGAACCAGAATCCTC-1368

D G T V T Q R D G Y G F I Y L L P F S R T R I L

ATCGAAGACACCAGATACTCCGACGGCGGCGACCTCGACGACGACGCACTCGCAGCAGCATCCCAAGACTAC-1440

I E D T R Y S D G G D L D D D A L A A A S Q D Y

GCAAGACAACAAGGCTGGACCGGTGCAGAAGTCAGAAGAGAAAGAGGCATCCTCCCAATCGCACTCGCACAC-1512

A R Q Q G W T G A E V R R E R G I L P I A L A H

GACGCAGCAGGTTTCTGGGCAGACCACGCAGAAGGTCCAGTCCCAGTCGGTCTCAGAGCAGGCTTCTTCCAC-1584

D A A G F W A D H A E G P V P V G L R A G F F H

CCAGTCACCGGCTACTCCCTCCCATACGCAGCACAAGTCGCAGACGTCGTCGCAGGTCTCTCCGGTCCACCA-1556

P V T G Y S L P Y A A Q V A D V V A G L S G P P

GGCACCGACGCACTCAGAGGTGCAATCAGAGACTACGCAATCGACAGAGCAAGAAGAGACAGATTCCTCAGA-1728

G T D A L R G A I R D Y A I D R A R R D R F L R

CTCCTCAACAGAATGCTCTTCAGAGGCTGCGCACCAGACAGAAGATACACCCTCCTCCAAAGACTCTACAGA-1800

L L N R M L F R G C A P D R R Y T L L Q R L Y R

ATGCCACACGGTCTCATCGAAAGATTCTACGCAGGTAGACTCTCCGTCGCAGACCAACTCAGAATCGTCACC-1872

M P H G L I E R F Y A G R L S V A D Q L R I V T

GGCAAGCCACCAATCCCACTCGGCACCGCAATCAGATGCCTCCCAGAAAGACCACTCCTCAAGGAAAACGCA-1994

G K P P I P L G T A I R C L P E R P L L K E N A

Redesign phytoene desaturase gene (crtZ) and beta carotene '-hydroxylase gene (crtZ) in the secondary coccus according to the plant-preference password) nucleotide sequence, and be combined into associating gene C rtIcrtZ.

The overlapping extension PCR method of synthetic employing of CrtIcrtZ gene carries out pcr amplification in conjunction with the high temperature resistant polysaccharase of high-fidelity.Nucleotide sequence is cloned and measured to gene in intestinal bacteria, be 2034bp Nucleotide from codon ATG to termination codon TAA length.Insert the preceding paragraph in 1485～1545 positions of gene and cut sequence automatically:

CAACTCCTCAACTTCGACCTCCTCAAACTCGCA

GGCGACGTCGAATCCAACCCAGGCCCA。

The Nucleotide and the aminoacid sequence of complete synthesis crtIcrtZ gene are as follows:

ATGAACGCACACTCCCCAGCAGCAAAGACCGCAATCGTCATCGGTGCAGGTTTCGGTGGTCTCGCACTCGCA--?72

M N A H S P A A K T A I V I G A G F G G L A L A

ATCAGACTCCAATCCGCAGGCATCGCAACCACCCTCGTCGAGGCAAGAGACAAGCCAGGCGGTAGAGCATAC--144

I R L Q S A G I A T T L V E A R D K P G G R A Y

GTCTGGCACGACCAAGGCCACGTCTTCGACGCAGGTCCAACCGTCATCACCGACCCAGACGCACTCAAGGAA--216

V W H D Q G H V F D A G P T V I T D P D A L K ECTCTGGGCACTCACCGGTCAAGACATGGCAAGAGACGTCACCCTCATGCCAGTCTCCCCATTCTACAGACTC--288?L W A L T G Q D?M A R D V T L M P V S P F Y R LATGTGGCCAGGTGGCAAGGTCTTCGACTACGTCAACGAGGCAGACCAACTCGAAAGACAAATCGCACAATTC--360?M W P G G K V F D Y V N E A D Q L E R Q I A Q FAACCCAGACGACCTCGAAGGTTACAGAAGATTCAGAGACTACGCAGAGGAGGTCTACCAGGAGGGCTACGTC--432?N P D D L E G Y R R F R D Y A E E V Y Q E G Y VAAACTCGGCACCGTCCCATTCCTCAAGCTCGGTCAAATGCTCAAGGCAGCACCAGCACTCATGAAACTCGAA--504?K L G T V P F L K L G Q M L K A A P A L M K L EGCATACAACTCCGTCCACGCAAAGGTCGCAACCTTCATCAAGGACCCATACCTCAGACAGGCATTCTCCTAC--576?A Y N S V H A K V A T F I K D P Y L R Q A F S YCACACCCTCCTCGTCGGTGGTAATCCATTCTCCACCTCCTCCATCTACGCACTCATCCACGCACTCGAAAGA--648?H T L L V G G N P F S T S S I Y A L I H A L E RAGAGGTGGTGTCTGGTTCGCAAAGGGTGGCACCAACCAGCTCGTCGCAGGTATGGTCGCACTCTTCGAGAGA--720?R G G V W F A K G G T N Q L V A G M V A L F E RCCAGGTGGTCAAATGATGCTCAACGCAAAGGTCGCAAGAATCGAAACCGAGGGTGCAAGAACCACCGGTGTC--792?P G G Q M M L N A K V A R I E T E G A R T T G VACCCTCGCAGACGGTAGATCCCTCAGAGCAGACATGGTCGCATCCAACGGTGACGTCATGCACAACTACAGA--864?T L A D G R S L R A D M V A S N G D V M H N Y RGACCTCCTCGGTCACACCGCAAGAGGTCAATCCAGAGCAAAGTCCCTCGACAGAAAGAGATGGTCCATGTCC--936?D L L G H T A R G Q S R A K S L D R K R W S M SCTCTTCGTCCTCCACTTCGGTCTCAGAGAAGCACCAAAGGACATCGCACACCACACCATCCTCTTCGGTCCA-1008?L F V L H F G L R E A P K D I A H H T I L F G PAGATACAAGGAACTCGTCAACGAAATCTTCAAGGGTCCAAAACTCGCAGAGGACTTCTCCCTCTACCTCCAC-1080?R Y K E L V N E I F K G P K L A E D F S L Y L HTCCCCATGCACCACCGACCCAGACATGGCACCACCAGGCATGTCCACCCACTACGTCCTCGCACCAGTCCCA-1152?S P C T T D P D M A P P G M S T H Y V L A P V PCACCTCTCCAGAGCAGAAATCGACTGGGCAGTCGAAGGCCCAAGATACGCAGACAGAATCCTCGCATTCCTC-1224?H L S R A E I D W A V E G P R Y A D R I L A F LGAAGAAAGACTCATCCCAAACCTCAGAGCAAACCTCACCAGAACCAGAATCTTCACCCCAGCAGACTTCGCA-1296?E E R L I P N L R A N L T R T R I F T P A D F ATCCGAACTCAACGCACACCACGGCTCCGCATTCTCCGTCGAACCAATCCTCACCCAATCCGCATGGTTCAGA-1368?S E L N A H H G S A F S V E P I L T Q S A W F RCCACACAACAGAGACAAGACCATCAGAAACTTCTACCTCGTCGGTGCAGGTACTCACCCAGGTGCAGGTATC-1440?P H N R D K T I R N F Y L V G A G T H P G A G ICCAGGTGTCGTCGGTTCCGCAAAGGCAACCGCACAAGTCATGCTCCAACTCCTCAACTTCGACCTCCTCAAA-1512?P G V V G S A K A T A Q V M L Q L L N F D L L KCTCGCAGGCGACGTCGAATCCAACCCAGGCCCAATGACCAACTTCCTCATCGTCGTCGCAACCGTCCTCGTC-1584?L A G D V E S N P G P M T N F L I V V A T V L VATGGAACTCACCGCATACTCCGTCCACAGATGGATCATGCACGGTCCACTCGGCTGGGGCTGGCACAAATCC-1656?M E L T A Y S V H R W I M H G P L G W G W H K SCACCACGAAGAACACGACCACGCACTCGAAAAAAACGACCTCTACGGTCTCGTCTTCGCAGTCATCGCAACC-1728?H H E E H D H A L E K N D L Y G L V F A V I A TGTCCTCTTCACCGTCGGTTGGATCTGGGCACCAGTCCTCTGGTGGATCGCACTCGGTATGACCGTCTACGGT-1800?V L F T V G W I W A P V L W W I A L G M T V Y GCTCATCTACTTCGTCCTCCACGACGGTGTCGTCCACCAAAGATGGCCATTCAGATACATCCCAAGAAAGGGC-1872

L I Y F V L H D G V V H Q R W P F R Y I P R K G

TACGCAAGAAGACTCTACCAAGCACACAGACTCCACCACGCAGTCGAAGGCAGAGACCACTGCGTCTCCTTC-1944

Y A R R L Y Q A H R L H H A V E G R D H C V S F

GGCTTCATCTACGCACCACCAGTCGATAAACTCAAAGAAGACCTCAAGACCTCCGGCGTCCTCAGAGCAGAA-2016

G F I Y A P P V D K L K E D L K T S G V L R A E

GCAGAAGAAAGAACCTAA-2034

A E E R T

Redesign the nucleotide sequence of phytoene synthase gene in the secondary coccus (crtB) and ridge ox base ridge base pyrophosphate synthetase gene (crtE) according to the plant-preference password, and form associating gene crtBcrtE.

The overlapping extension PCR method of synthetic employing of crtBcrtE gene carries out pcr amplification in conjunction with the high temperature resistant polysaccharase of high-fidelity.Nucleotide sequence is cloned and measured to gene in intestinal bacteria, be 1848bp Nucleotide from codon ATG to termination codon TAA length.Insert the preceding paragraph in 912～972 positions of gene and cut sequence automatically: CAACTCCTCAACTTCGACCTCCTCAAACTCGCAGGCGACGTCGAATCCAACCCAGG CCCA.

The Nucleotide and the aminoacid sequence of complete synthesis crtBcrtE gene are as follows:

ATGTCCGACCTCGTCCTCACCTCCACCGAGGCCATCACCCAGGGCTCCCAATCCTTCGCCACCGCAGCCAAA--?72

M S D L V L T S T E A I T Q G S Q S F A T A A K

CTCATGCCACCAGGCATCAGAGACGACACCGTCATGCTCTACGCCTGGTGCAGACACGCAGACGACGTCATC--144

L M P P G I R D D T V M L Y A W C R H A D D V I

GACGGTCAGGCACTCGGCTCCAGACCAGAGGCAGTCAACGACCCACAGGCCAGACTCGACGGTCTCAGAGCC--216

D G Q A L G S R P E A V N D P Q A R L D G L R A

GACACCCTCGCAGCACTCCAGGGTGACGGTCCAGTCACTCCACCATTCGCAGCACTCAGAGCAGTCGCAAGA--288

D T L A A L Q G D G P V T P P F A A L R A V A R

AGACACGACTTCCCACAGGCATGGCCAATGGACCTCATCGAGGGCTTCGCAATCGACGTTGAGGCAAGAGAC--360

R H D F P Q A W P M D L I E G F A I D V E A R D

TACAGAACCCTCGACGACGTCCTCGAATACTCCTACCACGTCGCAGGCATCCTCGGTGTCATGATGGCCAGA--432

Y R T L D D V L E Y S Y H V A G I L G V M M A R

GTCATGGGTGTCAGAGACCACCCAGTCCTCGACAGAGCCTGCGACCTCGGTCTCGCCTTCCAGCTCACCCAC--504

V M G V R D H P V L D R A C D L G L A F Q L T H

ATCGCAAGAGACGTCATCGACTACGCAAGAATCGGCAGATGCTACCTCCCAGGCGACTGGCTCGACCACGCA--576

I A R D V I D Y A R I G R C Y L P G D W L D H A

GGCGCAAGAGTCGATGGTCCAGTCCCATCCCCAGAACTCTACACCGTCATCCTCAGACTCCTCGACGCAGCA--648

G A R V D G P V P S P E L Y T V I L R L L D A A

GAGCCATACTACCCATCCGCAAGAGTCGGTCTCGCAGACCTCCCACCAAGATGCGCATGGTCCATCCCAGCA--720

E P Y Y P S A R V G L A D L P P R C A W S I P A

GCATTCAGAATCTACAGAGCAATCGGTCTCCCAATCAGAAAGGGTGGTCCAGAGGCATACAGACAAAGAATC--792

A F R I Y R A I G L P I R K G G P E A Y R Q R I

TCCACCTCCAAGGCAGCAAAGATCGGTCTCCTCGGCATCGGTGGTTGGGACGTCGCAGACCACGCTTGCAGA--864

S T S K A A K I G L L G I G G W D V A D H A C R

GGCTCCGGTGTCTCCAGACAAGACCTCTGGACCAGACCACACCACGCACAACTCCTCAACTTCGACCTCCTC--936

G S G V S R Q D L W T R P H H A Q L L N F D L L

AAACTCGCAGGCGACGTCGAATCCAACCCAGGCCCAATGAGAAGAGACGTCAACCCAATCCACGCAACCCTC--1008

K L A G D V E S N P G P M R R D V N P I H A T L

CTCCAAACCAGAATCGAAGAAATCGCACAAGGCTTCGGTGCAGTCCTCCAACCACTCGGTGCAGCAATGGCA--1080

L Q T R I E E I A Q G F G A V L Q P L G A A M A

GCACTCTCCTCCGGTAAGAGATTCAGAGGTATGCTCATGCTCCTCGCAGCAGAAGCATCCGGTGGTGTCTGC--1152

A L S S G K R F R G M L M L L A A E A S G G V C

GACACCATCGTCGATGCAGCCTGCGCAGTCGAAATGGTCCACGCAGCATCCCTCATCTTCGACGACCTCCCA--1224

D T I V D A A C A V E M V H A A S L I F D D L P

TGTATGGACGACGCAGGTCTCAGAAGAGGTCAACCAGCAACCCACGTCGCACACGGTGAATCCAGAGCAGTC--1296

C M D D A G L R R G Q P A T H V A H G E S R A V

CTCGGTGGCATCGCACTCATCACCGAAGCAATGGCACTCCTCGCAGGTGCAAGAGGTGCATCCGGTACTGTC--1368

L G G I A L I T E A M A L L A G A R G A S G T V

AGAGCACAACTCGTCAGAATCCTCTCCAGATCCCTCGGTCCACAAGGTCTCTGCGCAGGTCAAGACCTCGAC--1440

R A Q L V R I L S R S L G P Q G L C A G Q D L D

CTCCACGCAGCAAAGAACGGTGCAGGTGTCGAACAAGAACAAGACCTCAAAACCGGTGTCCTCTTCATCGCA--1512

L H A A K N G A G V E Q E Q D L K T G V L F I A

GGTCTCGAAATGCTCGCAGTCATCAAAGAGTTCGACGCAGAAGAACAAACCCAAATGATCGACTTCGGTAGA--1584

G L E M L A V I K E F D A E E Q T Q M I D F G R

CAACTCGGTAGAGTCTTCCAATCCTACGACGACCTCCTCGAAGTCGTCGGTGATCAAGCAGCACTCGGTAAA--1656

Q L G R V F Q S Y D D L L E V V G D Q A A L G K

GACACCGGTAGAGACGCAGCAGCACCAGGTCCAAGAAGAGGTCTCCTCGCAGTCTCCGACCTCCAAAAGGTC--1728

D T G R D A A A P G P R R G L L A V S D L Q K V

TCCAGACAATACGAAGCATCCAGAGCACAACTCGAAGGTATGGTCGGTTCCAAGAGACTCCAAGCACCAGAA--1800

S R Q Y E A S R A Q L E G M V G S K R L Q A P E

ATCGAAGCACTCCTCGAAAGAGTCCTCCCATACGCAGCAAGAGCATAA--1848

I E A L L E R V L P Y A A R A

The present invention utilizes advanced method for synthesizing gene, and 6 genes that synthesizing astaxanthin is necessary synthesize and clone by the method for amplification in vitro.The synthetic gene has the following advantages:

(1) with 6 gene crtW of astaxanthin synthetic in the secondary coccus, crtZ, crtY, crtI, crtB and crtE are genetic background, all adopt the plant-preference password, Nucleotide is transformed, noted simultaneously the inner conventional Nucleotide restriction endonuclease sites of gene is eliminated.

(2) 6 genes are combined in twos an associating gene, be crtWcrtY, crtIcrtZ, crtBcrtE adds the preceding paragraph in the junction of gene and gene and cuts sequence automatically: CAACTCCTCAACTTCGACCTCCTCAAACTCGCAGGCGACGTCGAATCCAACCCAGG CCCA.When the associating gene is expressed in plant, be divided into two, produce two protein with complete function.The synthetic construction step that reduces Expression element of associating gene makes polygenic Plant Transformation become simple and easy to do.

(3) 6 genes of synthetic have very strong function, can produce astaxanthin in lacking the intestinal bacteria of these genes, the bacterium colony express red.

Description of drawings

The design of primers of Fig. 1 .crtWcrtY gene fragment 1 (initial to 1008 sites) from 5 ' ATG

Fig. 2 .crtWcrtY gene fragment 2 (from 1008 sites to 3 ' TAA termination 1997) design of primers.

The design of primers of Fig. 3 .crtIcrtZ gene fragment 1 (initial to 1080 sites) from 5 ' ATG.

The design of primers of Fig. 4 .crtIcrtZ gene fragment 2 (from 1080 sites to 3 ' TAA stop 2034).

The design of primers of Fig. 5 .crtBcrtE gene fragment 1 (initial to 936 sites) from 5 ' ATG.

The design of primers of Fig. 6 .crtBcrtE gene fragment 2 (from 936 sites to 3 ' TAA stop 1848).

The building-up process of Fig. 7 .crtWcrtY gene.Earlier synthetic 1008bp (1) and 990bp (3) band connect into 1997bp (2) by PCR with two fragments.

The building-up process of Fig. 8 .crtIcrtZ gene.Earlier synthetic 1080bp (1) and 954bp (4) band connect into 2034bp (3) by PCR with two fragments.

The building-up process of Fig. 9 .crtBcrtE gene.Earlier synthetic 936bp (3) and 912bp (4) band connect into 1848bp (1) by PCR with two fragments.

Figure 10. 6 gene crtIcrtZ of astaxanthin synthetic, crtBcrtE, crtWcrtY are at expression in escherichia coli, and red bacterium colony is for transforming the intestinal bacteria of these genes.

The chemosynthesis of embodiment 1 crtWcrtY gene

According to design oligonucleotides synthetic primer illustrated in figures 1 and 2, primer length is 60-90bp, and overlap is 20-30bp between two primers, and the Tm value is 64-80 ℃.Oligonucleolide primers synthesizes commercialization, and is synthetic by Shanghai biotechnology branch office.

Utilize high temperature-resisting DNA polymerase eXTaq (the Japanese TAKANA company) synthetic gene of high-fidelity, all primers are added laggard performing PCR amplification, middle primer consumption is 5-10ng, and both sides primer consumption is 100-200ng, and the pcr amplification condition is 94 ℃, 30s; 60-72 ℃, 30s; 72 ℃, 1-2min.Carry out 25-35 circulation altogether.

The crtWcrtY gene divides two sections to synthesize:

First section begins to 1008 from the gene codon ATG, designs 16 primers altogether.The nucleotide sequence of primer is:

HCE1：

GGT，CTC，CTC，CTC，CCA，GTC，ATC，GTC，ACC，GTC，TAC，GCA，CTC，ATC，CTC，

GGT，GAC，AGA，TGG，ATG，TAC，GTC，GTC，TTC，TGG，CCA，CTC，CCA，TCC，ATC

HCE3：

ACT，TCG，ACC，ACG，GTG，GTC，CAG，TCA，GAT，GGT，ACG，CAA，GAT，TCA，TCG，

GTA，CTT，ACT，TCG，GTT，GGA，GAG，AAG，GTC，TCC，TCC，TCC，CAG，TCA，TCG

HCE5：

TCT，CCT，GGA，GAA，AGA，TGA，TCG，TCA，AGC，ACA，TGG，CAC，ACC，ACA，GAC，

ACG，CAG，GTA，CTG，ACG，ACG，ACC，CAG，ACT，TCG，ACC，ACG，GTG，GTC，CAG

HCE7：

GTC，GTC，CCA，GGT，AGA，CCA，AGA，GCA，AAC，GCA，GCA，ATG，GGT，CAA，CTC，

GTC，CTC，TGG，CTC，TAC，GCA，GGT，TTC，TCC，TGG，AGA，AAG，ATG，ATC，GTC

HCE9：

ACT，TCC，TCG，GTC，TCA，CCT，GGC，TCT，CCG，TCG，GTC，TCT，TCA，TCA，TCG，

CAC，ACG，ACG，CAA，TGC，ACG，GTT，CCG，TCG，TCC，CAG，GTA，GAC，CAA，GAG

HCE11：

GTC，CAC，GCA，CTC，TGG，TTC，CTC，GAC，GCA，GCA，GCA，CAC，CCA，ATC，CTC，

GCA，GTC，GCA，AAC，TTC，CTC，GGT，CTC，ACC，TGG，CTC

HCE13：

ACC，GCA，ACC，TCC，CTC，ATC，GTC，TCC，GGT，GGT，ATC，ATC，GCA?GCA，TGG，

CTC，GCA，CTC，CAC，GTC，CAC，GCA，CTC，TGG，TTC，CTC

HCE15：

AGG，ATC，CAA，CAA，TGT，CCG，CAC，ACG，CAC，TCC，CAA，AAG，CAG，ACC，TCA，

CCG，CAA，CCT，CCC，TCA，TCG，TC

HCE2：

GGG，AAT，GCG，TCG，TGA，CCT，GGT，CTG，TGT，GGG，AGC，CAG，GTG，CCG，AAG，

ACG，AAG，AGT，TGG，ATG，GAT，GCG，AGG，ATG，GTA，GGG，AGT，GGC，CAG，AAG

HCE4：

CGA，AGT，GGA，AGC，AGG，TGA，GGA，GGG，AGA，CTG，GGT，CGG，AGA，TTC，TGG，

AGG，ATC，TTG，CGT，TGT，GTC，TGT，CTG，GGA，ATG，CGT，CGT，GAC，CTG，GTC

HCE6：

GGT，TCT，GGT，GGA，TGG，GAG，TCT，CCA，CCA，AGG，GAC，GGT，TGG，GTG，GAG，

GTG，GTG，TTC，GTG，GTG，GTA，GCC，ACC，GAA，GTG，GAA，GCA，GGT，GAG，GAG

HCE8：

AT，TCG，ACG，TCG，CCT，GCG，AGT，TTG，AGG，AGG，TCG，AAG，TTG，AGG，AGT，

TGT，GCG，GTG，TCG，CCT，TTG，GTT，CTG，GTG，GAT，GGG，AGT，CTC

HCE10：

GCG，ATG，AGA，CCG，TTT，GCG，AGA，CCT，GCA，CCT，GCG，AGG，AGG，ACG，TCG，

TGG，GTC，ATT，GGG，CCT，GGG，TTG，GAT，TCG，ACG，TCG，CCT，GCG，AGT，TTG

HCE12：

CGG，ATG，GAC，CTG，CTG，CGT，GGT，CGA，GGA，GGA，GGA，CTC，TGA，GGT，CTG，

GTC，TTG，CTG，CTC，TGA，GTG，CGA，GTG，CGA，TGA，GAC，CGT，TTG，CGA，GAC

HCE14：

GAG，TGG，CTT，GAG，TCT，TGC，GAG，CCA，GTG，TGG，GGA，GAG，GTC，TGG，GTC，

GTG，GCA，GGA，CCA，GGT，GTG，ACC，TTC，GGA，TGG，ACC，TGC，TGC，GTG，GTC

HCE16：

GTT，GCG，AGT，CTT，CTT，GCG，TGT，CTT，GGG，AAT，CTG，ACT，TCT，TGG，TCT，

GGC，CAG，TTT，GCT，CTT，CTG，AGT，GGC，TTG，AGT，CTT，GCG，AGC

Second section begins to termination codon TAA from 1008 ATG of gene, designs 14 primers altogether.The nucleotide sequence of primer is:

HCF1：

CGA，CCT，CGA，CGA，CGA，CGC，ACT，CGC，AGC，AGC，ATC，CCA，AGA，CTA，CGC，

AAG，ACA，ACA，AGG，CTG，GAC，CGG，TGC，AGA，AGT，CAG，AAG，AGA，AAG，AGG

HCF3：

TTC，ATC，TAC，CTC，CTC，CCA，TTC，TCC，AGA，ACC，AGA，ATC，CTC，ATC，GAA，

GAC，ACC，AGA，TAC，TCC，GAC，GGC，GGC，GAC，CTC，GAC，GAC，GAC，GCA，CTC

HCF5：

AGA，CCA，CAC，GGT，GTC，CCA，AGA，CCA，GTC，ATC，ATG，GAC，GGC，ACC，GTC，

ACC，CAA，AGA，GAC，GGC，TAC，GGC，TTC，ATC，TAC，CTC，CTC，CCA，TTC，TCC

HCF7：

GCC，CAC，ACC，CAT，CCA，GAC，ACC，TCA，CCC，TCG，GCT，TCC，ACA，AGT，TCC，

TCG，GTG，TCG，AAA，TCG，AAA，CCG，ACA，GAC，CAC，ACG，GTG，TCC，CAA，GAC

HCF9：

ACG，CAC，AAG，GTG，CAA，CCC，TCT，CCT，GCG，GTA，CTA，GAA，TCG，AAG，CAG，

GCT，CCG，TCC，TCG，ACG，GCA，GAG，GCC，CAC，ACC，CAT，CCA，GAC，ACC

HCF11：

AGC，ACT，CGC，AGA，CGC，AGT，CGT，CAG，ATC，CGG，TGC，AGA，AAT，CAG，ATG，

GGA，CTC，CGA，CAT，CGC，ACT，CCT，CGA，CGC，ACA，AGG，TGC，AAC，CCT，CTC

HCF13：

CAA，GAC，ACG，CAA，GAA，GAC，TCG，CAA，CCG，GCT，ACG，GCT，CCC，TCG，ACG，

GTG，CAG，CAC，TCG，CAG，ACG，CAG，TCG，TC

HCF2：

GAC，TGG，ACC，TTC，TGC，GTG，GTC，TGC，CCA，GAA，ACC，TGC，TGC，GTC，GTG，

TGC，GAG，TGC，GAT，TGG，GAG，GAT，GCC，TCT，TTC，TCT，TCT，GAC，TTC，TGC

HCF4：

GAC，TTG，TGC，TGC，GTA，TGG，GAG，GGA，GTA，GCC，GGT，GAC，TGG，GTG，GAA，

GAA，GCC，TGC，TCT，GAG，ACC，GAC，TGG，GAC，TGG，ACC，TTC，TGC，GTG，GTC

HCF6：

TAG，TCT，CTG，ATT，GCA，CCT，CTG，AGT，GCG，TCG，GTG，CCT，GGT，GGA，CCG，

GAG，AGA，CCT，GCG，ACG，ACG，TCT，GCG，ACT，TGT，GCT，GCG，TAT，GGG，AGG

HCF8：

CA，GCC，TCT，GAA，GAG，CAT，TCT，GTT，GAG，GAG，TCT，GAG，GAA，TCT，GTC，

TCT，TCT，TGC，TCT，GTC，GAT，TGC，GTA，GTC，TCT，GAT，TGC，ACC，TCT，GAG

HCF10：

T，TCG，ATG，AGA，CCT，TGT，GGC，ATT，CTG，TAG，AGT，CTT，TGG，AGG，AGG，GTG，TAT，CTT，CTG，TCT，GGT，GCG，CAG，CCT，CTG，AAG，AGC，ATT，CTG，TTG

HCF12：

GGA，TTG，GTG，GCT，TGC，CGG，TGA，CGA，TTC，TGA，GTT，GGT，CTG，CGA，CGG，

AGA，GTC，TAC，CTG，CGT，AGA，ATC，TTT，CGA，TGA，GAC，CGT，GTG，GCA，TTC

HCF14：

TTG，AGC，TCT，TAT，GCG，TTT，TCC，TTG，AGG，AGT，GGT，CTT，TCT，GGG，AGG，

CAT，CTG，ATT，GCG，GTG，CCG，AGT，GGG，ATT，GGT，GGC，TTG，CCG，GTG，ACG

After two sections of genes are synthetic, the PCR product is mixed, carry out new round pcr amplification, two fragments are merged into an associating gene.The pcr amplification condition is 94 ℃, 30s; 62 ℃, 30s; 72 ℃, 2min 30s.Carry out 25-35 circulation altogether.Synthetic associating gene crtWcrtY total length is 1997bP.

The chemosynthesis of embodiment 2 crtIcrtZ genes

According to Fig. 3 and design oligonucleotides synthetic primer shown in Figure 4, primer length is 60-90bp, and overlap is 20-30bp between two primers, and the Tm value is 64-80 ℃.

The crtIcrtZ gene divides two sections to synthesize:

First section begins to 1080 from the gene codon ATG, designs 16 primers altogether.The nucleotide sequence of primer is:

HCC1：

TTC，CTC，AAG，CTC，GGT，CAA，ATG，CTC，AAG，GCA，GCA，CCA，GCA，CTC，ATG，

AAA，CTC，GAA，GCA，TAC，AAC，TCC，GTC，CAC，GCA，AAG，GTC，GCA，ACC，TTC

HCC3：

AGA，AGA，TTC，AGA，GAC，TAC，GCA，GAG，GAG，GTC，TAC，CAG，GAG，GGC，TAC，

GTC，AAA，CTC，GGC，ACC，GTC，CCA，TTC，CTC，AAG，CTC，GGT，CAA，ATG，CTC

HCC5：

GTC，AAC，GAG，GCA，GAC，CAA，CTC，GAA，AGA，CAA，ATC，GCA，CAA，TTC，AAC，

CCA，GAC，GAC，CTC，GAA，GGT，TAC，AGA，AGA，TTC，AGA，GAC，TAC，GCA，GAG

HCC7：

ACG，TCA，CCC，TCA，TGC，CAG，TCT，CCC，CAT，TCT，ACA，GAC，TCA，TGT，GGC，

CAG，GTG，GCA，AGG，TCT，TCG，ACT，ACG，TCA，ACG，AGG，CAG，ACC，AAC，TCG

HCC9：

AAC，CGT，CAT，CAC，CGA，CCC，AGA，CGC，ACT，CAA，GGA，ACT，CTG，GGC，ACT，

CAC，CGG，TCA，AGA，CAT，GGC，AAG，AGA，CGT，CAC，CCT，CAT，GCC，AGT，CTC

HCC11：

GCA，AGA，GAC，AAG，CCA，GGC，GGT，AGA，GCA，TAC，GTC，TGG，CAC，GAC，CAA，

GGC，CAC，GTC，TTC，GAC，GCA，GGT，CCA，ACC，GTC，ATC，ACC，GAC，CCA，GAC

HCC13：

AGG，TTT，CGG，TGG，TCT，CGC，ACT，CGC，AAT，CAG，ACT，CCA，ATC，CGC，AGG，

CAT，CGC，AAC，CAC，CCT，CGT，CGA，GGC，AAG，AGA，CAA，GCC，AGG，CGG，TAG

HCC15：

AGG，ATC，CAT，GGA，CGC，ACA，CTC，CCC，AGC，AGC，AAA，GAC，CGC，AAT，CGT，

CAT，CGG，TGC，AGG，TTT，CGG，TGG，TCT，CGC，ACT，C

HCC2：

GAG，AAT，GGA，TTA，CCA，CCG，ACG，AGG，AGG，GTG，TGG，TAG，GAG，AAT，GCC，

TGT，CTG，AGG，TAT，GGG，TCC，TTG，ATG，AAG，GTT，GCG，ACC，TTT，GCG，TGG

HCC4：

ACC，CTT，TGC，GAA，CCA，GAC，ACC，ACC，TCT，TCT，TTC，GAG，TGC，GTG，GAT，

GAG，TGC，GTA，GAT，GGA，GGA，GGT，GGA，GAA，TGG，ATT，ACC，ACC，GAC，GAG

HCC6：

TGA，GCA，TCA，TTT，GAC，CAC，CTG，GTC，TCT，CGA，AGA，GTG，CGA，CCA，TAC，

CTG，CGA，CGA，GCT，GGT，TGG，TGC，CAC，CCT，TTG，CGA，ACC，AGA，CAC，CAC

HCC8：

TAC，CGT，CTG，CGA，GGG，TGA，CAC，CGG，TGG，TTC，TTG，CAC，CCT，CGG，TTT，

CGA，TTC，TTG，CGA，CCT，TTG，CGT，TGA，GCA，TCA，TTT，GAC，CAC，CTG，GTC

HCC10：

ACC，GAG，GAG，GTC，TCT，GTA，GTT，GTG，CAT，GAC，GTC，ACC，GTT，GGA，TGC，

GAC，CAT，GTC，TGC，TCT，GAG，GGA，TCT，ACC，GTC，TGC，GAG，GGT，GAC，ACC

HCC12：

ACG，AAG，AGG，GAC，ATG，GAC，CAT，CTC，TTT，CTG，TCG，AGG，GAC，TTT，GCT，

CTG，GAT，TGA，CCT，CTT，GCG，GTG，TGA，CCG，AGG，AGG，TCT，CTG，TAG，TTG

HCC14：

ATC，TTG，GAC，CGA，AGA，GGA，TGG，TGT，GGT，GTG，CGA，TGT，CCT，TTG，GTG，

CTT，CTC，TGA，GAC，CGA，AGT，GGA，GGA，CGA，AGA，GGG，ACA，TGG，ACC，ATC

HCC16：

GG，AGG，TAG，AGG，GAG，AAG，TCC，TCT，GCG，AGT，TTT，GGA，CCC，TTG，AAG，ATT，TCG，TTG，ACG，AGT，TCC，TTG，TAT，CTT，GGA，CCG，AAG，AGG，ATG，GTG

Second section from 1080 of genes to the TAA termination codon, design 16 primers altogether.The nucleotide sequence of primer is:

HCD1：

GCA，ACC，GCA，CAA，GTC，ATG，CTC，CAA，CTC，CTC，AAC，TTC，GAC，CTC，CTC，

AAA，CTC，GCA，GGC，GAC，GTC，GAA，TCC，AAC，CCA，GGC，CCA，ATG，ACC，AAC

HCD3：

ACT，TCT，ACC，TCG，TCG，GTG，CAG，GTA，CTC，ACC，CAG，GTG，CAG，GTA，TCC，

CAG，GTG，TCG，TCG，GTT，CCG，CAA，AGG，CAA，CCG，CAC，AAG，TCA，TGC，TCC，

HCD5：

CTC，CGT，CGA，ACC，AAT，CCT，CAC，CCA，ATC，CGC，ATG，GTT，CAG，ACC，ACA，

CAA，CAG，AGA，CAA，GAC，CAT，CAG，AAA，CTT，CTA，CCT，CGT，CGG，TGC，AGG

HCD7：

AGA，ACC，AGA，ATC，TTC，ACC，CCA，GCA，GAC，TTC，GCA，TCC，GAA，CTC，AAC，

GCA，CAC，CAC，GGC，TCC，GCA，TTC，TCC，GTC，GAA，CCA，ATC，CTC，ACC

HCD9：

TAC，GCA，GAC，AGA，ATC，CTC，GCA，TTC，CTC，GAA，GAA，AGA，CTC，ATC，CCA，

AAC，CTC，AGA，GCA，AAC，CTC，ACC，AGA，ACC，AGA，ATC，TTC，ACC，CCA，GCA

HCD11：

GCA，CCA，GTC，CCA，CAC，CTC，TCC，AGA，GCA，GAA，ATC，GAC，TGG，GCA，GTC，

GAA，GGC，CCA，AGA，TAC，GCA，GAC，AGA，ATC，CTC，GCA，TTC

HCD13：

ATG，CAC，CAC，CGA，CCC，AGA，CAT，GGC，ACC，ACC，AGG，CAT，GTC，CAC，CCA，

CTA，CGT，CCT，CGC，ACC，AGT，CCC，ACA，CCT，CTC，C

HCD15：53bp

CAG，AGG，ACT，TCT，CCC，TCT，ACC，TCC，ACT，CCC，CAT，GCA，CCA，CCG，ACC，

CAG，ACA，TG

HCD2：

ATG，ATC，CAT，CTG，TGG，ACG，GAG，TAT，GCG，GTG，AGT，TCC，ATG，ACG，AGG，

ACG，GTT，GCG，ACG，ACG，ATG，AGG，AAG，TTG，GTC，ATT，GGG，CCT，GGG，TTG

HCD4：

TTT，TTC，GAG，TGC，GTG，GTC，GTG，TTC，TTC，GTG，GTG，GGA，TTT，GTG，CCA，

GCC，CCA，GCC，GAG，TGG，ACC，GTG，CAT，GAT，CCA，TCT，GTG，GAC，GGA，GTA

HCD6：

CCA，GAT，CCA，ACC，GAC，GGT，GAA，GAG，GAC，GGT，TGC，GAT，GAC，TGC，GAA，

GAC，GAG，ACC，GTA，GAG，GTC，GTT，TTT，TTC，GAG，TGC，GTG，GTC，GTG，TTC

HCD8：

TCG，TGG，AGG，ACG，AAG，TAG，ATG，AGA，CCG，TAG，ACG，GTC，ATA，CCG，AGT，

GCG，ATC，CAC，CAG，AGG，ACT，GGT，GCC，CAG，ATC，CAA，CCG，ACG，GTG，AAG

HCD10：

GAG，TCT，TCT，TGC，GTA，GCC，CTT，TCT，TGG，GAT，GTA，TCT，GAA，TGG，CCA，

TCT，TTG，GTG，GAC，GAC，ACC，GTC，GTG，GAG，GAC，GAA，GTA，GAT，GAG

HCD12：

AGA，TGA，AGC，CGA，AGG，AGA，CGC，AGT，GGT，CTC，TGC，CTT，CGA，CTG，CGT，

GGT，GGA，GTC，TGT，GTG，CTT，GGT，AGA，GTC，TTC，TTG，CGT，AGC，CCT，TTC

HCD14：

G，ACG，CCG，GAG，?GTC，TTG，AGG，TCT，TCT，TTG，AGT，TTA，TCG，ACT，GGT，GG

T，GCG，TAG，ATG，?AAG，CCG，AAG，GAG，ACG，CAG

HCD16：

G，TTG，GTG，ACC，TTA，GGT，TCT，TTC，TTC，TGC，TTC，TGC，TCT，GAG，GAC，GCC，GGA，GGT，CTT，GAG，GTC

After two sections of genes are synthetic, the PCR product is mixed, carry out new round pcr amplification, two fragments are merged into an associating gene.The pcr amplification condition is 94 ℃, 30s; 62 ℃, 30s; 72 ℃, 2min 30s.Carry out 25-35 circulation altogether.Synthetic associating gene crtIcrtE total length is 2034bP.

The chemosynthesis of embodiment 3 crtBcrtE genes

According to Fig. 5 and design oligonucleotides synthetic primer shown in Figure 6, primer length is 60-90bp, and overlap is 20-30bp between two primers, and the Tm value is 64-80 ℃.

The crtBcrtE gene divides two sections to synthesize:

First section begins to 936 from the gene codon ATG, designs 14 primers altogether.The nucleotide sequence of primer is:

HCA1：

TCC，TAC，CAC，GTC，GCA，GGC，ATC，CTC，GGT，GTC，ATG，ATG，GCC，AGA，GTC，

ATG，GGT，GTC，AGA，GAC，CAC，CCA，GTC，CTC，GAC，AGA，GCC，TGC，GAC，CTC

HCA3：

CTC，ATC，GAG，GGC，TTC，GCA，ATC，GAC，GTT，GAG，GCA，AGA，GAC，TAC，AGA，

ACC，CTC，GAC，GAC，GTC，CTC，GAA，TAC，TCC，TAC，CAC，GTC，GCA，GGC，ATC

HCA5：

CTC，CAC，CAT，TCG，CAG，CAC，TCA，GAG，CAG，TCG，CAA，GAA，GAC，ACG，ACT，

TCC，CAC，AGG，CAT，GGC，CAA，TGG，ACC，TCA，TCG，AGG，GCT，TCG，CAA，TCG

HCA7：

ACA，GGC，CAG，ACT，CGA，CGG，TCT，CAG，AGC，CGA，CAC，CCT，CGC，AGC，ACT，

CCA，GGG，TGA，CGG，TCC，AGT，CAC，TCC，ACC，ATT，CGC，AGC，ACT，CAG

HCA9：

TGC，AGA，CAC，GCA，GAC，GAC，GTC，ATC，GAC，GGT，CAG，GCA，CTC，GGC，TCC，

AGA，CCA，GAG，GCA，GTC，AAC，GAC，CCA，CAG，GCC，AGA，CTC，GAC，GGT，CTC

HCA11：

CCT，TCG，CCA，CCG，CAG，CCA，AAC，TCA，TGC，CAC，CAG，GCA，TCA，GAG，ACG，

ACA，CCG，TCA，TGC，TCT，ACG，CCT，GGT，GCA，GAC，ACG，CAG，ACG，ACG，TCA

HCA13：

AGG，ATC，CAA，CAA，TGT，CCG，ACC，TCG，TCC，TCA，CCT，CCA，CCG，AGG，CCA，

TCA，CCC，AGG，GCT，CCC，AAT，CCT，TCG，CCA，CCG，CAG，CCA，AAC

HCA2：

GTA，GCA，TCT，GCC，GAT，TCT，TGC，GTA，GTC，GAT，GAC，GTC，TCT，TGC，GAT，

GTG，GGT，GAG，CTG，GAA，GGC，GAG，ACC，GAG，GTC，GCA，GGC，TCT，GTC，GAG

HCA4：90bp

TAG，AGT，TCT，GGG，GAT，GGG，ACT，GGA，CCA，TCG，ACT，CTT，GCG，CCT，GCG，

TGG，TCG，AGC，CAG，TCG，CCT，GGG，AGG，TAG，CAT，CTG，CCG，ATT，CTT，GCG

HCA6：

GCG，AGA，CCG，ACT，CTT，GCG，GAT，GGG，TAG，TAT，GGC，TCT，GCT，GCG，TCG，

AGG，AGT，CTG，AGG，ATG，ACG，GTG，TAG，AGT，TCT，GGG，GAT，GGA，CTG，G

HCA8：

GGA，GAC，CGA，TTG，CTC，TGT，AGA，TTC，TGA，ATG，CTG，CTG，GGA，TGG，ACC，

ATG，CGC，ATC，TTG，GTG，GGA，GGT，CTG，CGA，GAC，CGA，CTC，TTG，CGG，ATG

HCA10：

CGA，TCT，TTG，CTG，CCT，TGG，AGG，TGG，AGA，TTC，TTT，GTC，TGT，ATG，CCT，

CTG，GAC，CAC，CCT，TTC，TGA，TTG，GGA，GAC，CGA，TTG，CTC，TGT，AGA，TTC

HCA12：

TGT，CTG，GAG，ACA，CCG，GAG，CCT，CTG，CAA，GCG，TGG，TCT，GCG，ACG，TCC，

CAA，CCA，CCG，ATG，CCG，AGG，AGA，CCG，ATC，TTT，GCT，GCC，TTG，GAG，GTG

HCA14：

GAG，GAG，GTC，GAA，GTT，GAG，GAG，TTG，TGC，GTG，GTG，TGG，TCT，GGT，CCA，

GAG，GTC，TTG，TCT，GGA，GAC，ACC，GGA，GCC，TC

Second section from 936 of genes to the TAA termination codon, design 14 primers altogether.The nucleotide sequence of primer is:

HCB1：

CGC，ACT，CAT，CAC，CGA，AGC，AAT，GGC，ACT，CCT，CGC，AGG，TGC，AAG，AGG，

TGC，ATC，CGG，TAC，TGT，CAG，AGC，ACA，ACT，CGT，CAG，AAT，CCT，CTC，CAG

HCB3：

GTC，TCA，GAA，GAG，GTC，AAC，CAG，CAA，CCC，ACG，TCG，CAC，ACG，GTG，AAT，

CCA，GAG，CAG，TCC，TCG，GTG，GCA，TCG，CAC，TCA，TCA，CCG，AAG，CAA，TGG

HCB5：

CGC，AGT，CGA，AAT，GGT，CCA，CGC，AGC，ATC，CCT，CAT，CTT，CGA，CGA，CCT，

CCC，ATG，TAT，GGA，CGA，CGC，AGG，TCT，CAG，AAG，AGG，TCA，ACC，AGC，AAC

HCB7：

GTA，TGC，TCA，TGC，TCC，TCG，CAG，CAG，AAG，CAT，CCG，GTG，GTG，TCT，GCG，

ACA，CCA，TCG，TCG，ATG，CAG，CCT，GCG，，CAG，TCG，AAA，TGG，TCC，ACG，CAG

HCB9：

CTT，CGG，TGC，AGT，CCT，CCA，ACC，ACT，CGG，TGC，AGC，AAT，GGC，AGC，ACT，

CTC，CTC，CGG，TAA，GAG，ATT，CAG，AGG，TAT，GCT，CAT，GCT，CCT，CGC，AGC

HCB11：

ATG，AGA，AGA，GAC，GTC，AAC，CCA，ATC，CAC，GCA，ACC，CTC，CTC，CAA，ACC，

AGA，ATC，GAA，GAA，ATC，GCA，CAA，GGC，TTC，GGT，GCA，GTC，CTC，CAA，CCA

HCB13：

CAC，AAC，TCC，TCA，ACT，TCG，ACC，TCC，TCA，AAC，TCG，CAG，GCG，ACG，TCG，

AAT，CCA，ACC，CAG，GCC，CAA，TGA，GAA，GAG，ACG，TCA，ACC，CAA，TC

HCB2：

TGC，ACC，GTT，CTT，TGC，TGC，GTG，GAG，GTC，GAG，GTC，TTG，ACC，TGC，GCA，

GAG，ACC，TTG，TGG，ACC，GAG，GGA，TCT，GGA，GAG，GAT，TCT，GAC，GAG，TTG

HCB4：

ACT，GCG，AGC，ATT，TCG，AGA，CCT，GCG，ATG，AAG，AGG，ACA，CCG，GTT，TTG，

AGG，TCT，TGT，TCT，TGT，TCG，ACA，CCT，GCA，CCG，TTC，TTT，GCT，GCG，TGG

HCB6：

A，CTC，TAC，CGA，GTT，GTC，TAC，CGA，AGT，CGA，TCA，TTT，GGG，TTT，GTT，C

TT，CTG，CGT，CGA，ACT，CTT，TGA，TGA，CTG，CGA，GCA，TTT，CGA，GAC，CTG

HCB8：

CCG，GTG，TCT，TTA，CCG，AGT，GCT，GCT，TGA，TCA，CCG，ACG，ACT，TCG，AGG，

AGG，TCG，TCG，TAG，GAT，TGG，AAG，ACT，CTA，CCG，AGT，TGT，CTA，CCG，AAG

HCB10：

GAC，CTT，TTG，GAG，GTC，GGA，GAC，TGC，GAG，GAG，ACC，TCT，TCT，TGG，ACC，

TGG，TGC，TGC，TGC，GTC，TCT，ACC，GGT，GTC，TTT，ACC，GAG，TGC，TGC，TTG

HCB12：

GTG，CTT，GGA，GTC，TCT，TGG，AAC，CGA，CCA，TAC，CTT，CGA，GTT，GTG，CTC，

TGG，ATG，CTT，CGT，ATT，GTC，TGG，AGA，CCT，TTT，GGA，GGT，CGG，AGA，CTG

HCB14：

TT，GAG，CTC，TTA，TGC，TCT，TGC，TGC，GTA，TGG，GAG，GAC，TCT，TTC，GAG，

GAG，TGC，TTC，GAT，TTC，TGG，TGC，TTG，GAG，TCT，CTT，GGA，ACC

After two sections of genes are synthetic, the PCR product is mixed, carry out new round pcr amplification, two fragments are merged into an associating gene.The pcr amplification condition is 94 ℃, 30s; 62 ℃, 30s; 72 ℃, 2min 30s.Carry out 25-35 circulation altogether.Synthetic associating gene crtBcrtE total length is 1848bP.

The Function Identification of embodiment 4 synthetic genes.

Utilize PCR method to add BamHI and SalI restriction enzyme site at the two ends of synthetic gene crtWcrtY, add SalI and HindIII restriction enzyme site at the two ends of synthetic gene crtIcrtZ, add EcoRI+SacI and KpnI restriction enzyme site at the two ends of synthetic gene crtBcrtE.The crtWcrtY gene is inserted the pBluescripsMCS carrier, and (three Room, biological center, academy of agricultural sciences, Shanghai make up, 1998), insert the crtIcrtZ gene again at SalI and HindIII restriction enzyme site afterwards, 4 placed in-line genes are cut the back with BamHI and KpnI enzyme insert the corresponding restriction enzyme site of pCAMBIA1301 carrier (available from U.S. CAMBIA company), cut the crtBcrtE gene with EcoRI and KpnI enzyme again, insert the corresponding restriction enzyme site of this carrier, constitute the placed in-line associating gene of 6 genes.

In order to verify the function of gene, with 6 gene constructed under prokaryotic promoter control, transformed into escherichia coli allows them at expression in escherichia coli.After cutting tandem gene with BamHI and SacI enzyme, (three Room, biological center, academy of agricultural sciences, Shanghai make up to insert pYP2001,1998) corresponding restriction enzyme site, make up the expression system of 6 tandem genes, chemical method transformed into escherichia coli DH5 α, intestinal bacteria are uniformly coated on the flat board that contains the kantlex substratum, cultivate after 12 hours for 37 ℃, observe the colour-change (Figure 10) of bacterium colony, the intestinal bacteria that red bacterium colony occurs show that gene has been transformed in the intestinal bacteria, and given expression to activated protein therein, make the intestinal bacteria synthesizing astaxanthin.

Claims

1, by ridge ox base ridge ox base pyrophosphate synthetase gene (crtE) from secondary coccus; Phytoene synthase gene (crtB); Phytoene desaturase gene (crtI); Lycopene beta cyclase gene (crtY); β-Hu Luobusu '-hydroxylase gene (crtZ) and β-Hu Luobusu ketolase gene (crtW) is characterized in that becoming leading astaxanthin biological synthetic crtW crtY gene and crtI crtZ and crtB crtE associating gene by aforementioned six kinds of genetic modifications.

2, as follows according to described leading astaxanthin biological synthetic crtWcrtY gene nucleotide of claim 1 and aminoacid sequence:

ATGTCCGCACACGCACTCCCAAAAGCAGACCTCACCGCAACCTCCCTCATCGTCTCCGGTGGTATCATCGCA--?72

M S A H A L P K A D L T A T S L I V S G G I I A

GCATGGCTCGCACTCCACGTCCACGCACTCTGGTTCCTCGACGCAGCAGCACACCCAATCCTCGCAGTCGCA--144

A W L A L H V H A L W F L D A A A H P I L A V A

AACTTCCTCGGTCTCACCTGGCTCTCCGTCGGTCTCTTCATCATCGCACACGACGCAATGCACGGTTCCGTC--216

N F L G L T W L S V G L F I I A H D A M H G S V

GTCCCAGGTAGACCAAGAGCAAACGCAGCAATGGGTCAACTCGTCCTCTGGCTCTACGCAGGTTTCTCCTGG--288

V P G R P R A N A A M G Q L V L W L Y A G F S W

AGAAAGATGATCGTCAAGCACATGGCACACCACAGACACGCAGGTACTGACGACGACCCAGACTTCGACCAC--360

R K M I V K H M A H H R H A G T D D D P D F D H

GGTGGTCCAGTCAGATGGTACGCAAGATTCATCGGTACTTACTTCGGTTGGAGAGAAGGTCTCCTCCTCCCA--432

G G P V R W Y A R F I G T Y F G W R E G L L L P

GTCATCGTCACCGTCTACGCACTCATCCTCGGTGACAGATGGATGTACGTCGTCTTCTGGCCACTCCCATCC--504

V I V T V Y A L I L G D R W M Y V V F W P L P S

ATCCTCGCATCCATCCAACTCTTCGTCTTCGGCACCTGGCTCCCACACAGACCAGGTCACGACGCATTCCCA--576

I L A S I Q L F V F G T W L P H R P G H D A F P

GACAGACACAACGCAAGATCCTCCAGAATCTCCGACCCAGTCTCCCTCCTCACCTGCTTCCACTTCGGTGGC--648

D R H N A R S S R I S D P V S L L T C F H F G G

TACCACCACGAACACCACCTCCACCCAACCGTCCCTTGGTGGAGACTCCCATCCACCAGAACCAAAGGCGAC--720

Y H H E H H L H P T V P W W R L P S T R T K G D

ACCGCACAACTCCTCAACTTCGACCTCCTCAAACTCGCAGGCGACGTCGAATCCAACCCAGGCCCAATGACC--792

T A Q L L N F D L L K L A G D V E S N P G P M T

CACGACGTCCTCCTCGCAGGTGCAGGTCTCGCAAACGGTCTCATCGCACTCGCACTCAGAGCAGCAAGACCA--864

H D V L L A G A G L A N G L I A L A L R A A R P

GACCTCAGAGTCCTCCTCCTCGACCACGCAGCAGGTCCATCCGAAGGTCACACCTGGTCCTGCCACGACCCA--936

D L R V L L L D H A A G P S E G H T W S C H D P

GACCTCTCCCCACACTGGCTCGCAAGACTCAAGCCACTCAGAAGAGCAAACTGGCCAGACCAAGAAGTCAGA-1008

D L S P H W L A R L K P L R R A N W P D Q E V R

TTCCCAAGACACGCAAGAAGACTCGCAACCGGCTACGGCTCCCTCGACGGTGCAGCACTCGCAGACGCAGTC-1080

F P R H A R R L A T G Y G S L D G A A L A D A V

GTCAGATCCGGTGCAGAAATCAGATGGGACTCCGACATCGCACTCCTCGACGCACAAGGTGCAACCCTCTCC-1152

V R S G A E I R W D S D I A L L D A Q G A T L S

TGCGGTACTAGAATCGAAGCAGGCTCCGTCCTCGACGGCAGAGGCCCACACCCATCCAGACACCTCACCCTC-1224

C G T R I E A G S V L D G R G P H P S R H L T L

GGCTTCCACAAGTTCCTCGGTGTCGAAATCGAAACCGACAGACCACACGGTGTCCCAAGACCAGTCATCATG-1296

G F H K F L G V E I E T D R P H G V P R P V I M

GACGGCACCGTCACCCAAAGAGACGGCTACGGCTTCATCTACCTCCTCCCATTCTCCAGAACCAGAATCCTC-1368

D G T V T Q R D G Y G F I Y L L P F S R T R I L

ATCGAAGACACCAGATACTCCGACGGCGGCGACCTCGACGACGACGCACTCGCAGCAGCATCCCAAGACTAC-1440

I E D T R Y S D G G D L D D D A L A A A S Q D Y

GCAAGACAACAAGGCTGGACCGGTGCAGAAGTCAGAAGAGAAAGAGGCATCCTCCCAATCGCACTCGCACAC-1512

A R Q Q G W T G A E V R R E R G I L P I A L A H

GACGCAGCAGGTTTCTGGGCAGACCACGCAGAAGGTCCAGTCCCAGTCGGTCTCAGAGCAGGCTTCTTCCAC-1584

D A A G F W A D H A E G P V P V G L R A G F F H

CCAGTCACCGGCTACTCCCTCCCATACGCAGCACAAGTCGCAGACGTCGTCGCAGGTCTCTCCGGTCCACCA-1556

P V T G Y S L P Y A A Q V A D V V A G L S G P P

GGCACCGACGCACTCAGAGGTGCAATCAGAGACTACGCAATCGACAGAGCAAGAAGAGACAGATTCCTCAGA-1728

G T D A L R G A I R D Y A I D R A R R D R F L R

CTCCTCAACAGAATGCTCTTCAGAGGCTGCGCACCAGACAGAAGATACACCCTCCTCCAAAGACTCTACAGA-1800

L L N R M L F R G C A P D R R Y T L L Q R L Y R

ATGCCACACGGTCTCATCGAAAGATTCTACGCAGGTAGACTCTCCGTCGCAGACCAACTCAGAATCGTCACC-1872

M P H G L I E R F Y A G R L S V A D Q L R I V T

GGCAAGCCACCAATCCCACTCGGCACCGCAATCAGATGCCTCCCAGAAAGACCACTCCTCAAGGAAAACGCA-1994

G K P P I P L G T A I R C L P E R P L L K E N A

3, as follows according to described leading astaxanthin biological synthetic crtIcrtZ gene nucleotide of claim 1 and aminoacid sequence:

ATGAACGCACACTCCCCAGCAGCAAAGACCGCAATCGTCATCGGTGCAGGTTTCGGTGGTCTCGCACTCGCA--?72

M N A H S P A A K T A I V I G A G F G G L A L A

ATCAGACTCCAATCCGCAGGCATCGCAACCACCCTCGTCGAGGCAAGAGACAAGCCAGGCGGTAGAGCATAC--144

I R L Q S A G I A T T L V E A R D K P G G R A Y

GTCTGGCACGACCAAGGCCACGTCTTCGACGCAGGTCCAACCGTCATCACCGACCCAGACGCACTCAAGGAA--216

V W H D Q G H V F D A G P T V I T D P D A L K E

CTCTGGGCACTCACCGGTCAAGACATGGCAAGAGACGTCACCCTCATGCCAGTCTCCCCATTCTACAGACTC--288

L W A L T G Q D M A R D V T L M P V S P F Y R L

ATGTGGCCAGGTGGCAAGGTCTTCGACTACGTCAACGAGGCAGACCAACTCGAAAGACAAATCGCACAATTC--360

M W P G G K V F D Y V N E A D Q L E R Q I A Q FAACCCAGACGACCTCGAAGGTTACAGAAGATTCAGAGACTACGCAGAGGAGGTCTACCAGGAGGGCTACGTC--432?N P D D L E G Y R R F R D Y A E E V Y Q E G Y VAAACTCGGCACCGTCCCATTCCTCAAGCTCGGTCAAATGCTCAAGGCAGCACCAGCACTCATGAAACTCGAA--504?K L G T V P F L K L G Q M L K A A P A L M K L EGCATACAACTCCGTCCACGCAAAGGTCGCAACCTTCATCAAGGACCCATACCTCAGACAGGCATTCTCCTAC--576?A Y N S V H A K V A T F I K D P Y L R Q A F S YCACACCCTCCTCGTCGGTGGTAATCCATTCTCCACCTCCTCCATCTACGCACTCATCCACGCACTCGAAAGA--648?H T L L V G G N P F S T S S I Y A L I H A L E RAGAGGTGGTGTCTGGTTCGCAAAGGGTGGCACCAACCAGCTCGTCGCAGGTATGGTCGCACTCTTCGAGAGA--720?R G G V W F A K G G T N Q L V A G M V A L F E RCCAGGTGGTCAAATGATGCTCAACGCAAAGGTCGCAAGAATCGAAACCGAGGGTGCAAGAACCACCGGTGTC--792?P G G Q M M L N A K V A R I E T E G A R T T G VACCCTCGCAGACGGTAGATCCCTCAGAGCAGACATGGTCGCATCCAACGGTGACGTCATGCACAACTACAGA--864?T L A D G R S L R A D M V A S N G D V M H N Y RGACCTCCTCGGTCACACCGCAAGAGGTCAATCCAGAGCAAAGTCCCTCGACAGAAAGAGATGGTCCATGTCC--936?D L L G H T A R G Q S R A K S L D R K R W S M SCTCTTCGTCCTCCACTTCGGTCTCAGAGAAGCACCAAAGGACATCGCACACCACACCATCCTCTTCGGTCCA-1008?L F V L H F G L R E A P K D I A H H T I L F G PAGATACAAGGAACTCGTCAACGAAATCTTCAAGGGTCCAAAACTCGCAGAGGACTTCTCCCTCTACCTCCAC-1080?R Y K E L V N E I F K G P K L A E D F S L Y L HTCCCCATGCACCACCGACCCAGACATGGCACCACCAGGCATGTCCACCCACTACGTCCTCGCACCAGTCCCA-1152?S P C T T D P D M A P P G M S T H Y V L A P V PCACCTCTCCAGAGCAGAAATCGACTGGGCAGTCGAAGGCCCAAGATACGCAGACAGAATCCTCGCATTCCTC-1224?H L S R A E I D W A V E G P R Y A D R I L A F LGAAGAAAGACTCATCCCAAACCTCAGAGCAAACCTCACCAGAACCAGAATCTTCACCCCAGCAGACTTCGCA-1296?E E R L I P N L R A N L T R T R I F T P A D F ATCCGAACTCAACGCACACCACGGCTCCGCATTCTCCGTCGAACCAATCCTCACCCAATCCGCATGGTTCAGA-1368?S E L N A H H G S A F S V E P I L T Q S A W F RCCACACAACAGAGACAAGACCATCAGAAACTTCTACCTCGTCGGTGCAGGTACTCACCCAGGTGCAGGTATC-1440?P H N R D K T I R N F Y L V G A G T H P G A G ICCAGGTGTCGTCGGTTCCGCAAAGGCAACCGCACAAGTCATGCTCCAACTCCTCAACTTCGACCTCCTCAAA-1512?P G V V G S A K A T A Q V M L Q L L N F D L L KCTCGCAGGCGACGTCGAATCCAACCCAGGCCCAATGACCAACTTCCTCATCGTCGTCGCAACCGTCCTCGTC-1584?L A G D V E S N P G P M T N F L I V V A T V L VATGGAACTCACCGCATACTCCGTCCACAGATGGATCATGCACGGTCCACTCGGCTGGGGCTGGCACAAATCC-1656?M E L T A Y S V H R W I M H G P L G W G W H K SCACCACGAAGAACACGACCACGCACTCGAAAAAAACGACCTCTACGGTCTCGTCTTCGCAGTCATCGCAACC-1728?H H E E H D H A L E K N D L Y G L V F A V I A TGTCCTCTTCACCGTCGGTTGGATCTGGGCACCAGTCCTCTGGTGGATCGCACTCGGTATGACCGTCTACGGT-1800?V L F T V G W I W A P V L W W I A L G M T V Y GCTCATCTACTTCGTCCTCCACGACGGTGTCGTCCACCAAAGATGGCCATTCAGATACATCCCAAGAAAGGGC-1872?L I Y F V L H D G V V H Q R W P F R Y I P R K GTACGCAAGAAGACTCTACCAAGCACACAGACTCCACCACGCAGTCGAAGGCAGAGACCACTGCGTCTCCTTC-1944?Y A R R L Y Q A H R L H H A V E G R D H C V S FGGCTTCATCTACGCACCACCAGTCGATAAACTCAAAGAAGACCTCAAGACCTCCGGCGTCCTCAGAGCAGAA-2016

G F I Y A P P V D K L K E D L K T S G V L R A E

GCAGAAGAAAGAACCTAA-2034

A E E R T

4, as follows according to described leading astaxanthin biological synthetic crtBcrtE gene nucleotide of claim 1 and aminoacid sequence:

ATGTCCGACCTCGTCCTCACCTCCACCGAGGCCATCACCCAGGGCTCCCAATCCTTCGCCACCGCAGCCAAA--?72

M S D L V L T S T E A I T Q G S Q S F A T A A K

CTCATGCCACCAGGCATCAGAGACGACACCGTCATGCTCTACGCCTGGTGCAGACACGCAGACGACGTCATC--144

L M P P G I R D D T V M L Y A W C R H A D D V I

GACGGTCAGGCACTCGGCTCCAGACCAGAGGCAGTCAACGACCCACAGGCCAGACTCGACGGTCTCAGAGCC--216

D G Q A L G S R P E A V N D P Q A R L D G L R A

GACACCCTCGCAGCACTCCAGGGTGACGGTCCAGTCACTCCACCATTCGCAGCACTCAGAGCAGTCGCAAGA--288

D T L A A L Q G D G P V T P P F A A L R A V A R

AGACACGACTTCCCACAGGCATGGCCAATGGACCTCATCGAGGGCTTCGCAATCGACGTTGAGGCAAGAGAC--360

R H D F P Q A W P M D L I E G F A I D V E A R D

TACAGAACCCTCGACGACGTCCTCGAATACTCCTACCACGTCGCAGGCATCCTCGGTGTCATGATGGCCAGA--432

Y R T L D D V L E Y S Y H V A G I L G V M M A R

GTCATGGGTGTCAGAGACCACCCAGTCCTCGACAGAGCCTGCGACCTCGGTCTCGCCTTCCAGCTCACCCAC--504

V M G V R D H P V L D R A C D L G L A F Q L T H

ATCGCAAGAGACGTCATCGACTACGCAAGAATCGGCAGATGCTACCTCCCAGGCGACTGGCTCGACCACGCA--576

I A R D V I D Y A R I G R C Y L P G D W L D H A

GGCGCAAGAGTCGATGGTCCAGTCCCATCCCCAGAACTCTACACCGTCATCCTCAGACTCCTCGACGCAGCA--648

G A R V D G P V P S P E L Y T V I L R L L D A A

GAGCCATACTACCCATCCGCAAGAGTCGGTCTCGCAGACCTCCCACCAAGATGCGCATGGTCCATCCCAGCA--720

E P Y Y P S A R V G L A D L P P R C A W S I P A

GCATTCAGAATCTACAGAGCAATCGGTCTCCCAATCAGAAAGGGTGGTCCAGAGGCATACAGACAAAGAATC--792

A F R I Y R A I G L P I R K G G P E A Y R Q R I

TCCACCTCCAAGGCAGCAAAGATCGGTCTCCTCGGCATCGGTGGTTGGGACGTCGCAGACCACGCTTGCAGA--864

S T S K A A K I G L L G I G G W D V A D H A C R

GGCTCCGGTGTCTCCAGACAAGACCTCTGGACCAGACCACACCACGCACAACTCCTCAACTTCGACCTCCTC--936

G S G V S R Q D L W T R P H H A Q L L N F D L L

AAACTCGCAGGCGACGTCGAATCCAACCCAGGCCCAATGAGAAGAGACGTCAACCCAATCCACGCAACCCTC--1008

K L A G D V E S N P G P M R R D V N P I H A T L

CTCCAAACCAGAATCGAAGAAATCGCACAAGGCTTCGGTGCAGTCCTCCAACCACTCGGTGCAGCAATGGCA--1080

L Q T R I E E I A Q G F G A V L Q P L G A A M A

GCACTCTCCTCCGGTAAGAGATTCAGAGGTATGCTCATGCTCCTCGCAGCAGAAGCATCCGGTGGTGTCTGC--1152

A L S S G K R F R G M L M L L A A E A S G G V C

GACACCATCGTCGATGCAGCCTGCGCAGTCGAAATGGTCCACGCAGCATCCCTCATCTTCGACGACCTCCCA--1224

D T I V D A A C A V E M V H A A S L I F D D L P

TGTATGGACGACGCAGGTCTCAGAAGAGGTCAACCAGCAACCCACGTCGCACACGGTGAATCCAGAGCAGTC--1296

C M D D A G L R R G Q P A T H V A H G E S R A V

CTCGGTGGCATCGCACTCATCACCGAAGCAATGGCACTCCTCGCAGGTGCAAGAGGTGCATCCGGTACTGTC--1368

L G G I A L I T E A M A L L A G A R G A S G T V

AGAGCACAACTCGTCAGAATCCTCTCCAGATCCCTCGGTCCACAAGGTCTCTGCGCAGGTCAAGACCTCGAC--1440

R A Q L V R I L S R S L G P Q G L C A G Q D L D

CTCCACGCAGCAAAGAACGGTGCAGGTGTCGAACAAGAACAAGACCTCAAAACCGGTGTCCTCTTCATCGCA--1512

L H A A K N G A G V E Q E Q D L K T G V L F I A

GGTCTCGAAATGCTCGCAGTCATCAAAGAGTTCGACGCAGAAGAACAAACCCAAATGATCGACTTCGGTAGA--1584

G L E M L A V I K E F D A E E Q T Q M I D F G R

CAACTCGGTAGAGTCTTCCAATCCTACGACGACCTCCTCGAAGTCGTCGGTGATCAAGCAGCACTCGGTAAA--1656

Q L G R V F Q S Y D D L L E V V G D Q A A L G K

GACACCGGTAGAGACGCAGCAGCACCAGGTCCAAGAAGAGGTCTCCTCGCAGTCTCCGACCTCCAAAAGGTC--1728

D T G R D A A A P G P R R G L L A V S D L Q K V

TCCAGACAATACGAAGCATCCAGAGCACAACTCGAAGGTATGGTCGGTTCCAAGAGACTCCAAGCACCAGAA--1800

S R Q Y E A S R A Q L E G M V G S K R L Q A P E

ATCGAAGCACTCCTCGAAAGAGTCCTCCCATACGCAGCAAGAGCATAA--1848

I E A L L E R V L P Y A A R A

5, the synthetic method of leading astaxanthin biological synthetic crtWcrtY, crtIcrtZ and crtBcrtE gene, it is characterized in that adopting the high temperature resistant polysaccharase PCR of high-fidelity to increase, nucleotide sequence is cloned and surveyed to gene in intestinal bacteria, insert automatically cutting sequence different positions inserts cutting sequence automatically in the junction of crtWcrtY, crtIcrtZ and crtBcrtE gene

CAACTCCTCAACTTCGACCTCCTCAAACTCGCAGGCGACGTCGA

ATCCAACCCAGGCCCA synthesizes.

6, according to the synthetic method of the described leading astaxanthin biological synthetic gene of claim 5, it is characterized in that synthetic crtWcrtY gene, totally 1997 nucleotide bases are right from codon ATG to termination codon TAA, insert cutting sequence C AACTCCTCAACTTCGACCTCCTCAAACTCGCAGGCGACGTCGAATCCAACCCAGGC CCA automatically in 726～786 positions of gene.

7, according to the synthetic method of the described leading astaxanthin biological synthetic gene of claim 5, it is characterized in that synthetic crtIcrtZ gene, totally 2034 nucleotide bases are right from codon ATG to termination codon TAA, insert cutting sequence automatically in 1485～1545 positions of gene

CAACTCCTCAACTTCGACCTCCTCAAACTCGCA

GGCGACGTCGAATCCAACCCAGGCCCA。

8, according to the synthetic method of the described leading astaxanthin biological synthetic gene of claim 5, it is characterized in that synthetic crtBcrtE gene, totally 1848 nucleotide bases are right from codon ATG to termination codon TAA, insert one in 912～972 positions of gene and cut sequence C AACTCCTCAACTTCGACCTCCTCAAACTCGCAGGCGACGTCGAATCCAACCCAGGC CCA automatically.

9, according to the synthetic method of the described leading astaxanthin biological synthetic gene of claim 5, it is characterized in that crtWcrtY, crtIcrtZ, the two ends of crtBcrtE add BamHI and SalI restriction enzyme site respectively; SalI and HindIII restriction enzyme site; EcoRI+SacI and KpnI restriction enzyme site, the crtWcrtY gene is inserted the pBluescripsMCS carrier, SalI and HindIII restriction enzyme site are inserted the crtIcrtZ gene again, again four placed in-line genes are cut the back with BamHI and KpnI enzyme and insert the corresponding restriction enzyme site of pCAMBIA1301 carrier, cut the crtBcrtE gene with EcoRI and KpnI enzyme again, insert the corresponding restriction enzyme site of this carrier, constitute the placed in-line associating gene of 6 genes.

10, use 6 genes series connection of employing according to the described leading astaxanthin biological synthetic gene function of claim 1, be structured in prokaryotic promoter control down, transformed into escherichia coli, make them at expression in escherichia coli, after cutting tandem gene with BamHI and SacI enzyme, insert the corresponding restriction enzyme site of pYP2001, be built into the expression system of six tandem genes, given expression to activated protein and made the intestinal bacteria synthesizing astaxanthin.

11, according to the described leading astaxanthin biological synthetic gene of claim 1, it is characterized in that by crtW and crtY; CrtI and crtZ; CrtB and crtE are composed in series 3 associating genes, according to the preference password synthesizing ribonucleotide of plant.

12, leading according to claim 1 astaxanthin biological synthetic gene can and utilize in the plant production astaxanthin in the plant modification quality and use.